Troubleshooting

→ Works. Could mean that P1 adapter ligation failed and only fragments with P2 adapters on both ends are amplifying.

Start with leftover DNA from Greenbuls Plate 2 and re-shear to see if overshearing caused lack of P1 adapter to most fragments.

1) Pre-cleaned DNA (post-P1-ligation), 2) Cleaned DNA, 3) Sheared DNA

1st shearing step looks good - proceed to Blunt End Repair → PCR

1) 1μL template, 2) 5μL PCR product
→ Still no amplification. Sonication is not the issue.

Test Sunbirds Plate 1 template and replicate PCR, and check if failing templates will amplify with only P2 Primer

1&2) Sunbirds Plate 1, 3&4) Greenbuls Plate 1 (from re-sheared template)
→ PCR with good template still works. P2 primer only does not work at all

Re-prep Greenbuls Plate 1 using leftover DNA from plate

2014-07-31:

Digestion: RADIGEST on Tony

Ligation: RADLIG on Tony

Used adapters from original Berkeley plate (undiluted)

Clean up

Sonication: 8 cycles of 15 seconds on, 90 seconds off, High Power

Do not have have gel image for 2nd shearing attempt (4 additional cycles), but looked better than first. Because final attempt with additional 2 cycles did not look right (more large fragments), decided to continue with subsequent steps to avoid further sonication problems.

End Repair: NEBENDRP on John Block B

P2 Ligation: NEBLIGAT on John Block B

2014-08-01:

Size Selection: 40μL beads to remove large fragments (decreased amount to reduce loss of DNA), 25μL to remove small